Horm. Metab. Res. 7 (1975) 400-402
@ Georg Thieme Verlag Stuttgart
Evidence for the D-Ce11 of the Pancreas Secreting Somatostatin L. Orci, D. Baetens, M.P. Dubois and C. Rufener Institute of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland and I.N.R.A., Station de Physiologie de la Reproduction, Nol~zilly.France Summarv -~----..-By immunofluorescence, somatostatin-, glucagon- and insulin-containing cells are localized in serial sections of the pigeon pancreas. The distribution of the somatostatin im-
D-cell is responsible for the secretion of somatostatin. Key-Words: Somatostatin - D-Ce11 - Endocrine Pancreas Pigeon - Immunofluorescence Electron Microscopy
-
For immunofluorescence (indirect technique) (Coons, Leduc und Connolly 1955), serial sections were rehydrated after arid incubated with one of the followremoval of ing antiSera for one hour: rabbit anti-somatostatin
I
Introduction Somatostatin, a polypeptide extracted from the hypothalamus (Brazeau, Vale, Burgus, Ling, Butcher, Rivier und Guillemin 1973), was shown by immunofluorescence to be also present in specific cells of the pancreas (Luft, Efendic, Hökfelt, Johansson und Arimura 1974, Dubois 1975) and of the gastro-intestinal tract (Rufener, Dubois, Malaisse-Lagae und Orci in press). However, the ultrastructure of the anti-somatostatin fluorescent cell is entirely unknown at present. A candidate for being the somatostatin-producing cell seems to be the D-cell, since this cell, although previously supposed to secrete gastrin (Lomsky, Lange und Vortel 1969, Greider und McGuigan 197 1) is now without a precise function (Lotstra, von der Loo und Gepts 1974). Evidence for this hypothesis was seeked in the pancreas of the pigeon. In this species, D-cells (A, -cells) are particularly numerous (Roth 1968). According to Roth, the pigeon pancreas contains three types of islets as determined with the light microscope (Roth 1968): a) "light" islets, in which B-cells predominate and are surrounded by numerous D-cells; b) "dark" islets, containing no Bcells but composed of D (A,) and A (A2) cells; C) "mixed" islets formed by a majority of D- and A-cells with a few clusters bf B-cells.
I
Materials and Methods Pieces of the ventral, dorsal and splenic lobes of fresh pigeon fluorescence. Embedding was either in Epon or in paraffin.
Received: 16 M y 1975
Accepted: 16 June 1975
I Fig. 1. Three consecutive serial sections from dorsal lobe of pigeon pancreas. Section incubated with anti-somatostatin serum (la), with anti-glucagon serum ( I b ) and with anti-insulin serum(1c). In each picture the arrows point to mixed islets showing somatostatin-, glucagon- and insulincontaining cells respectively. The dark islet labelled with an arrow-head does not reveal insulin-containing cells (X 225). The horizontal bar represents 20 /im.
Downloaded by: Universite Laval. Copyrighted material.
-
Fig. 2. a. Part of a "dark" islet, composed of A- and D-cells. The A-cell granules possess two components: an inner core of high electron opacity and a peripheral mantle of lesser electron density. (X 13.000) b. Part of a "mixed" islet, constituted of A-, B- and D-cells. The B-cell granules possess irregular cores which frequently show a crystalloid appearance. (X 1 3 000). The horizontal bar represents 1
rabbit anti-gastrin (1:10), rabbit anti-glucagon (1:20) and guinea-pig anti-insulin (1:SO) diluted in phosphate buffer saline (PBS). After washing in PBS, the sections were further incubated with sheep antirabbit or anti-guinea pig gamma globulines conjugated with fluorescein isothiocyanate (FITC) (Pasteur Institute, Paris) for one hour, washed in PBS, counterstained with 0.1%Evans blue and mounted in glycerinPBS. Control sections were incubated with one of the antisera after adsorption with an excess of the corresponding antigen (cyclic somatostatin, 200 pg/ml; gastrin or glucagon, 50 pg(ml; insulin, 2 Ulml). In addition, incwbations were performed with anti-sornatostatin adsorbed with either insulin, glucagon or gastrin.
Fig. 3. Part of a "dark" islet showing several D-cells. The delta granules are usually of lower electron density than the alpha granules, with limiting membranes closely applied to the granulecores. The different cytoplasmic electron density of the cells illustrated here remains to be assessed (X 14.000). The horizontal bar represents 1 p.
Downloaded by: Universite Laval. Copyrighted material.
Evidence for the DCeU of the Pancreas Secreting Somatostatin
L. Orci, D. Baetens, M.P. Dubois and C. Rufener
Results and Discussion
Immunofluorescent cells to anti-somatostatin, antiglucagon and anti-insulin were found in islets of ventral, dorsal and splenic lobes. Immunofluorescent cells to anti-gastrin were not detected (Fig. 1 a-c). . In control sections, immunofluorescence was aboltshed after adsorption of the antiserum with the corresponding antigen. In sections incubated with anti-insulin or anti-glucagon, immunofluorescence w~s not s~ppressed by adsorption with cyclic somatost~tm. In~ultn, glucagon and gastrin did not interfere wlt~ the l~muno cytological binding of the somatostatm anhs~rum to the pancreatic cells. That the antisomatostatm flu~r. escence in certain cells could be due to a nonspeclfIc adsorption of circulating somatostatin on such cells. seems unlikely since immunofluorescent cells to anhsomatostatin were also found in monolayer cultures of rat endocrine pancreas after several washings in plasma-free synthetic media.
these cells correspond respectively to 0-, A- and Bcells. This leads to the conclusion that D-cells are most Iikely the cells responsible for the secretion of somatostatin or somatostatin-Iike peptide.·
In 1969 Hellman and Lernmark reported inhibition of insulin secretion by microdissected mouse islets in the presence of extracts of pigeon pancreas. This inhibitory effect was attributed to a secretory product of the AI cell (D-cell). The known insulin-inhibiting action of somatostatin would reconcile those findings with the present observations. The direct identification of the somatostatin-producing cells as D-cells awaits now the successful application of immunocytochemical techniques at the ultrastructural level. Acknowledgements
We are grateful to Dr. J. Rivier and Dr. R. GuiHemin for their generous gift of cyclic somatostatin. We are also very o~hged to Dr. W. Gepts for providing synthetic gastrm and antl-gastfln In serial sections, the three fluorescent cell types were serum, to Dr. R. Vnger for anti-glucagon serum and to Dr. P.H. distinct one from another by their respective distribu- Wright for anti-insulin serum. We wish to acknowledge the extion in light, dark and mixed islets (Fig. 1 a-c). The cellent technical assistance of Mrs. I. Fughster and A. Irnbarra. number of anti-somatostatin fluorescent cells was This work was supported by grants no 3.8081.72, 3.0310.73, 3.553.75, from the Fonds National Suisse de la Recherche much larger in the islets of the pigeon than in islets Scientifique and by grant no 74-4-445-35 from I.N.S.E.R.M. of mammalian pancreas (rat, dog, pig and man). IFrance).
At the ultrastructural level, A, Band D-cells were identified (Fig. 2 a-b; Fig. 3) on the basis of the characteristics of their respective secretory granules (Kobayashi and Fujita 1969). The number and disposition within the three islet types of anti-somatostatin, anti-glucagon and anti-insulin fluorescent cells, coupled with the ultrastructural observations, seem to allow us to propose that
A d den d u m: By using a peroxidase labeHing technique, we were recently able to identify ultrastructurally the somatostatincontaining ceH as the D-ceH (Rufener el al. submitted for publication).
Downloaded by: Universite Laval. Copyrighted material.
402
*Recently we were able to demonstrate anti-somatostatin fluorescent cells in the gastro-intestinal tract of the dog (Rufener et al. in press), of man, guinea pig, rat, mouse and pig (unpublished data). Similar rcsults wcre reported independently by Polak ct al. (1975).
References
8rauau, P., W. Vale, R. Burgus, N. Ling. M. 8utcher, J. Ri· monstration of gastrin in mammalian islets of Langerhans. vier, R. Guillemin: Hypothalamic polypeptide that inhibits Nature 223: 618-619 (1969) the secretion of immunoreactive pituitary growth hormone. Lotstra, F., W. van der Loo, W. Gepts: Are gastrin-ceHs preScience 179: 77-79 (1973) sent in mammalian pancreatic islets? Diabetologia 10: Coons, A.H., E.H. Leduc, J.M. Connolly: Studies on antibody 291-302 (1974) production. I. A method for the histochemical demonstra- Luft, R., S. Efendic, T. Hökfelt, o. Johansson, A. Arimura: tion of specific antibody and its application to a study of Immunohistochemical evidence for the localization of the hyperimmune rabbit. J.Exp.Med. 102: 49-63 (1955) somatostatin-Iikc immunoreactivity in a ceH population of Du bois, M.P.: Presence of immunoreactive somatostatin in the pancreatic islets. Medical Biology 52: 428-430 (1974) Polak, J.M., A. G.E. Pearse, L. Grimelius, S.R. Bloom, A. Ari. discrete cells of the endocrine pancreas. Proc.NatI.Acad. Sei. (V.S.A.) 72: 1340-1343 (1975) mura: Growth-hormone releasc-inhibiting hormone in gasGreider, M.H., J.E. McGuigan: CeHular localization of gastrin trointestinal and pancreatic D-cells. Lancet 7918: 1220in the human pancreas. Diabetes 20: 389-396 (1971) 1222 (1975) Hel/man, 8., A. Lernmark: A possible role of the pancreatic Roth, A.: Quantitative studies on the islets of Langerhans in QI- and Q2- cells as local regulators of insulin secretion. the pigeon. Acta anat. 69: 609-622 (1968) In: The structure and metabolism of the pancreatic islets, Rufener, c., M.P. Du bois, F. Malaisse.Lagae, L. Orei: Immuno-fluorescent reactivity to anti-somatostatin in the Falkmer, S., B. Hellman and I.B. Täljedal, Editors. Pergamon Press, Oxford 453-462 (1969) gastro-intestinal mucosa of the dog. Diabetologia (in press) Kobayashi, S., T. Fujita: Fine structure of mammalian and Rufener, C., M. Amherdt, M.P. Dubois, L. Orei: Vltrastructural avian pancreatic islets with special reference to D-cells immunocytochemicallocalization of somatostatin in D-cell, and nervous elements. Z.Zellforsch. 100: 340-363 (1969) of rat pancreatic monolayer culture (submitted for puhlica. Lomsky, R., F. Lange, V. Vortel: Immunohistochemical detion). Requests for reprints should be addressed to: L. Orci, Institute of Histology and Embryology, Vniversity of Geneva Medical School, CH-1211 Geneva 4 (Switzerland)
@ Georg Thieme Verlag Stuttgart
Evidence for the D-Ce11 of the Pancreas Secreting Somatostatin L. Orci, D. Baetens, M.P. Dubois and C. Rufener Institute of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland and I.N.R.A., Station de Physiologie de la Reproduction, Nol~zilly.France Summarv -~----..-By immunofluorescence, somatostatin-, glucagon- and insulin-containing cells are localized in serial sections of the pigeon pancreas. The distribution of the somatostatin im-
D-cell is responsible for the secretion of somatostatin. Key-Words: Somatostatin - D-Ce11 - Endocrine Pancreas Pigeon - Immunofluorescence Electron Microscopy
-
For immunofluorescence (indirect technique) (Coons, Leduc und Connolly 1955), serial sections were rehydrated after arid incubated with one of the followremoval of ing antiSera for one hour: rabbit anti-somatostatin
I
Introduction Somatostatin, a polypeptide extracted from the hypothalamus (Brazeau, Vale, Burgus, Ling, Butcher, Rivier und Guillemin 1973), was shown by immunofluorescence to be also present in specific cells of the pancreas (Luft, Efendic, Hökfelt, Johansson und Arimura 1974, Dubois 1975) and of the gastro-intestinal tract (Rufener, Dubois, Malaisse-Lagae und Orci in press). However, the ultrastructure of the anti-somatostatin fluorescent cell is entirely unknown at present. A candidate for being the somatostatin-producing cell seems to be the D-cell, since this cell, although previously supposed to secrete gastrin (Lomsky, Lange und Vortel 1969, Greider und McGuigan 197 1) is now without a precise function (Lotstra, von der Loo und Gepts 1974). Evidence for this hypothesis was seeked in the pancreas of the pigeon. In this species, D-cells (A, -cells) are particularly numerous (Roth 1968). According to Roth, the pigeon pancreas contains three types of islets as determined with the light microscope (Roth 1968): a) "light" islets, in which B-cells predominate and are surrounded by numerous D-cells; b) "dark" islets, containing no Bcells but composed of D (A,) and A (A2) cells; C) "mixed" islets formed by a majority of D- and A-cells with a few clusters bf B-cells.
I
Materials and Methods Pieces of the ventral, dorsal and splenic lobes of fresh pigeon fluorescence. Embedding was either in Epon or in paraffin.
Received: 16 M y 1975
Accepted: 16 June 1975
I Fig. 1. Three consecutive serial sections from dorsal lobe of pigeon pancreas. Section incubated with anti-somatostatin serum (la), with anti-glucagon serum ( I b ) and with anti-insulin serum(1c). In each picture the arrows point to mixed islets showing somatostatin-, glucagon- and insulincontaining cells respectively. The dark islet labelled with an arrow-head does not reveal insulin-containing cells (X 225). The horizontal bar represents 20 /im.
Downloaded by: Universite Laval. Copyrighted material.
-
Fig. 2. a. Part of a "dark" islet, composed of A- and D-cells. The A-cell granules possess two components: an inner core of high electron opacity and a peripheral mantle of lesser electron density. (X 13.000) b. Part of a "mixed" islet, constituted of A-, B- and D-cells. The B-cell granules possess irregular cores which frequently show a crystalloid appearance. (X 1 3 000). The horizontal bar represents 1
rabbit anti-gastrin (1:10), rabbit anti-glucagon (1:20) and guinea-pig anti-insulin (1:SO) diluted in phosphate buffer saline (PBS). After washing in PBS, the sections were further incubated with sheep antirabbit or anti-guinea pig gamma globulines conjugated with fluorescein isothiocyanate (FITC) (Pasteur Institute, Paris) for one hour, washed in PBS, counterstained with 0.1%Evans blue and mounted in glycerinPBS. Control sections were incubated with one of the antisera after adsorption with an excess of the corresponding antigen (cyclic somatostatin, 200 pg/ml; gastrin or glucagon, 50 pg(ml; insulin, 2 Ulml). In addition, incwbations were performed with anti-sornatostatin adsorbed with either insulin, glucagon or gastrin.
Fig. 3. Part of a "dark" islet showing several D-cells. The delta granules are usually of lower electron density than the alpha granules, with limiting membranes closely applied to the granulecores. The different cytoplasmic electron density of the cells illustrated here remains to be assessed (X 14.000). The horizontal bar represents 1 p.
Downloaded by: Universite Laval. Copyrighted material.
Evidence for the DCeU of the Pancreas Secreting Somatostatin
L. Orci, D. Baetens, M.P. Dubois and C. Rufener
Results and Discussion
Immunofluorescent cells to anti-somatostatin, antiglucagon and anti-insulin were found in islets of ventral, dorsal and splenic lobes. Immunofluorescent cells to anti-gastrin were not detected (Fig. 1 a-c). . In control sections, immunofluorescence was aboltshed after adsorption of the antiserum with the corresponding antigen. In sections incubated with anti-insulin or anti-glucagon, immunofluorescence w~s not s~ppressed by adsorption with cyclic somatost~tm. In~ultn, glucagon and gastrin did not interfere wlt~ the l~muno cytological binding of the somatostatm anhs~rum to the pancreatic cells. That the antisomatostatm flu~r. escence in certain cells could be due to a nonspeclfIc adsorption of circulating somatostatin on such cells. seems unlikely since immunofluorescent cells to anhsomatostatin were also found in monolayer cultures of rat endocrine pancreas after several washings in plasma-free synthetic media.
these cells correspond respectively to 0-, A- and Bcells. This leads to the conclusion that D-cells are most Iikely the cells responsible for the secretion of somatostatin or somatostatin-Iike peptide.·
In 1969 Hellman and Lernmark reported inhibition of insulin secretion by microdissected mouse islets in the presence of extracts of pigeon pancreas. This inhibitory effect was attributed to a secretory product of the AI cell (D-cell). The known insulin-inhibiting action of somatostatin would reconcile those findings with the present observations. The direct identification of the somatostatin-producing cells as D-cells awaits now the successful application of immunocytochemical techniques at the ultrastructural level. Acknowledgements
We are grateful to Dr. J. Rivier and Dr. R. GuiHemin for their generous gift of cyclic somatostatin. We are also very o~hged to Dr. W. Gepts for providing synthetic gastrm and antl-gastfln In serial sections, the three fluorescent cell types were serum, to Dr. R. Vnger for anti-glucagon serum and to Dr. P.H. distinct one from another by their respective distribu- Wright for anti-insulin serum. We wish to acknowledge the extion in light, dark and mixed islets (Fig. 1 a-c). The cellent technical assistance of Mrs. I. Fughster and A. Irnbarra. number of anti-somatostatin fluorescent cells was This work was supported by grants no 3.8081.72, 3.0310.73, 3.553.75, from the Fonds National Suisse de la Recherche much larger in the islets of the pigeon than in islets Scientifique and by grant no 74-4-445-35 from I.N.S.E.R.M. of mammalian pancreas (rat, dog, pig and man). IFrance).
At the ultrastructural level, A, Band D-cells were identified (Fig. 2 a-b; Fig. 3) on the basis of the characteristics of their respective secretory granules (Kobayashi and Fujita 1969). The number and disposition within the three islet types of anti-somatostatin, anti-glucagon and anti-insulin fluorescent cells, coupled with the ultrastructural observations, seem to allow us to propose that
A d den d u m: By using a peroxidase labeHing technique, we were recently able to identify ultrastructurally the somatostatincontaining ceH as the D-ceH (Rufener el al. submitted for publication).
Downloaded by: Universite Laval. Copyrighted material.
402
*Recently we were able to demonstrate anti-somatostatin fluorescent cells in the gastro-intestinal tract of the dog (Rufener et al. in press), of man, guinea pig, rat, mouse and pig (unpublished data). Similar rcsults wcre reported independently by Polak ct al. (1975).
References
8rauau, P., W. Vale, R. Burgus, N. Ling. M. 8utcher, J. Ri· monstration of gastrin in mammalian islets of Langerhans. vier, R. Guillemin: Hypothalamic polypeptide that inhibits Nature 223: 618-619 (1969) the secretion of immunoreactive pituitary growth hormone. Lotstra, F., W. van der Loo, W. Gepts: Are gastrin-ceHs preScience 179: 77-79 (1973) sent in mammalian pancreatic islets? Diabetologia 10: Coons, A.H., E.H. Leduc, J.M. Connolly: Studies on antibody 291-302 (1974) production. I. A method for the histochemical demonstra- Luft, R., S. Efendic, T. Hökfelt, o. Johansson, A. Arimura: tion of specific antibody and its application to a study of Immunohistochemical evidence for the localization of the hyperimmune rabbit. J.Exp.Med. 102: 49-63 (1955) somatostatin-Iikc immunoreactivity in a ceH population of Du bois, M.P.: Presence of immunoreactive somatostatin in the pancreatic islets. Medical Biology 52: 428-430 (1974) Polak, J.M., A. G.E. Pearse, L. Grimelius, S.R. Bloom, A. Ari. discrete cells of the endocrine pancreas. Proc.NatI.Acad. Sei. (V.S.A.) 72: 1340-1343 (1975) mura: Growth-hormone releasc-inhibiting hormone in gasGreider, M.H., J.E. McGuigan: CeHular localization of gastrin trointestinal and pancreatic D-cells. Lancet 7918: 1220in the human pancreas. Diabetes 20: 389-396 (1971) 1222 (1975) Hel/man, 8., A. Lernmark: A possible role of the pancreatic Roth, A.: Quantitative studies on the islets of Langerhans in QI- and Q2- cells as local regulators of insulin secretion. the pigeon. Acta anat. 69: 609-622 (1968) In: The structure and metabolism of the pancreatic islets, Rufener, c., M.P. Du bois, F. Malaisse.Lagae, L. Orei: Immuno-fluorescent reactivity to anti-somatostatin in the Falkmer, S., B. Hellman and I.B. Täljedal, Editors. Pergamon Press, Oxford 453-462 (1969) gastro-intestinal mucosa of the dog. Diabetologia (in press) Kobayashi, S., T. Fujita: Fine structure of mammalian and Rufener, C., M. Amherdt, M.P. Dubois, L. Orei: Vltrastructural avian pancreatic islets with special reference to D-cells immunocytochemicallocalization of somatostatin in D-cell, and nervous elements. Z.Zellforsch. 100: 340-363 (1969) of rat pancreatic monolayer culture (submitted for puhlica. Lomsky, R., F. Lange, V. Vortel: Immunohistochemical detion). Requests for reprints should be addressed to: L. Orci, Institute of Histology and Embryology, Vniversity of Geneva Medical School, CH-1211 Geneva 4 (Switzerland)
